Soybean rust, caused by the obligate pathogen Phakopsora pachyrhizi, is considered to be one of the most serious foliar diseases of soybean worldwide. To successfully isolate, culture, and multiply the pathogen, a detached-leaf assay previously developed was reevaluated. In a separate study, the effect of temperature and relative humidity (RH) was studied on urediniospore survival. Three susceptible genotypes, Williams 82, TGx 1485-1D, and Taki Hime, and two genotypes with resistance genes, PI462312 (Rpp3) and PI459025B (Rpp4), were used to study the detached-leaf assay; only Williams 82 was used for the urediniospore survival study. The detached-leaf assay using agar medium amended with BAP performed better in retarding leaf chlorosis than did the filter paper method. Among the three susceptible genotypes, detached leaves of Williams 82 had the lowest level of leaf chlorosis; they were found to be the most suitable in the detached-leaf assay for pathogen isolation, multiplication, and short-term preservation. Urediniospore survival was highly affected by temperature and RH. Urediniospores maintained at room temperature survived for 18 days when kept on infected leaves and up to 30 days in cryovials. In the RH trial, urediniospores harvested from infected leaves maintained at 85% RH (23–24°C) had the highest spore germination and longevity. P. pachyrhizi cultural techniques improvement and spore survival study will help in understanding the biology, host-plant relationship, and conditions that favor the infection and the survival of the pathogen.